JP7336573B2 - Light sheet microscope and sample observation method - Google Patents

Description

The present invention relates to a light sheet microscope and a sample observation method.

2. Description of the Related Art A light sheet microscope is known that irradiates a sample with sheet-like excitation light and detects detection light emitted from the sample along with irradiation of the excitation light (see, for example, Patent Document 1). In such a light sheet microscope, a container containing a sample is held by a holder, and when the sample is observed, the holder is moved or rotated to scan the irradiation position of the sample with excitation light.

Japanese Patent Publication No. 2006-509246

However, with the light sheet microscope as described above, the sample may shake during observation, and stable observation may not be possible. Moreover, since the scanning speed of the excitation light is determined by the moving speed of the holder, it is difficult to speed up the observation. Although it is conceivable to arrange the sample on the stage and move or rotate the stage, there are the same problems as in the case of moving or rotating the holder in terms of stability and speeding up.

SUMMARY OF THE INVENTION An object of the present invention is to provide a light sheet microscope and a sample observation method capable of achieving high-speed and stable observation.

The light sheet microscope of the present invention comprises an irradiation optical system for irradiating a sample with excitation light including a wavelength that excites the sample, a detection optical system for guiding detection light emitted from the sample along with the irradiation of the excitation light, and a detection a photodetector for detecting the detection light guided by the optical system, wherein the irradiation optical system includes a wavelength swept light source that outputs light whose wavelength changes with time as excitation light; Relay optics including a spectroscopic element that receives excitation light and emits the excitation light at an emission angle corresponding to the wavelength of the excitation light, and a cylindrical lens that allows the excitation light emitted from the spectroscopic element to enter at an incident angle corresponding to the emission angle. and a first objective lens for condensing the excitation light guided by the relay optical system and irradiating the sample with the sheet-shaped excitation light.

In this light sheet microscope, light whose wavelength changes with time is output from a wavelength swept light source as excitation light, and the excitation light output from the wavelength swept light source is emitted from a spectroscopic element at an emission angle corresponding to the wavelength. The emitted excitation light enters the cylindrical lens at an incident angle corresponding to the emitted angle. Thereby, the irradiation position of the sheet-like excitation light can be scanned with respect to the sample. As a result, since it is not necessary to move or rotate the sample during observation, it is possible to suppress the sample from shaking and to observe the sample stably. In addition, since the wavelength swept light source, spectroscopic element, and cylindrical lens are used to scan the irradiation position of the excitation light on the sample, the scanning speed of the excitation light on the sample can be increased. Therefore, according to this light sheet microscope, high-speed and stable observation can be realized.

In the light sheet microscope of the present invention, the spectral element may be a diffraction grating or a prism. According to these configurations, it is possible to remarkably achieve the above operational effect that high-speed and stable observation can be realized.

In the light sheet microscope of the present invention, the detection optical system includes the second objective lens into which the detection light is incident, and the focal position of the second objective lens in synchronization with changes in the wavelength of the excitation light output from the wavelength swept light source. and a variable focus position adjuster. According to this configuration, even when the irradiation position of the excitation light on the sample is scanned at high speed, the detection light can be detected with high accuracy.

In the light sheet microscope of the invention, the focus position adjuster may be a liquid lens. According to this configuration, even when the irradiation position of the excitation light on the sample is scanned at high speed, it is possible to detect the detection light with high accuracy.

In the light sheet microscope of the present invention, the wavelength swept light source is configured to switch the center wavelength of the excitation light among a plurality of center wavelengths, and the spectroscopic element is configured to switch the center wavelength of the excitation light output from the wavelength swept light source. may be rotated according to According to this configuration, the sample can be irradiated with excitation light having a plurality of center wavelengths.

In the light sheet microscope of the present invention, the illumination optical system has a plurality of wavelength swept light sources, and the plurality of wavelength swept light sources output light having different center wavelengths as excitation light, and the light is output from the plurality of wavelength swept light sources. The excitation light may enter the spectroscopic element at an incident angle according to its center wavelength. According to this configuration, the sample can be irradiated with excitation light having a plurality of center wavelengths.

The light sheet microscope of the present invention further comprises a moving mechanism for moving the wavelength swept light source, the spectroscopic element and the cylindrical lens along the optical axis of the cylindrical lens while maintaining the positional relationship between the spectroscopic element and the cylindrical lens. good too. According to this configuration, it is possible to adjust the position of the beam waist of the excitation light emitted from the first objective lens, that is, the formation position of the sheet-shaped excitation light in the irradiation direction of the excitation light to the sample.

The light sheet microscope of the present invention may further include a reflector that reflects the excitation light emitted from the first objective lens toward the sample. Further, the light sheet microscope of the present invention may further include a container in which the sample is placed, and the container may be provided with a reflecting section. According to this configuration, the sample can be irradiated with the sheet-like excitation light without being affected by the wall of the container.

In the light sheet microscope of the present invention, the detection optical system may have a second objective lens on which detection light is incident, and the optical axis of the second objective lens may be parallel to the optical axis of the first objective lens. This configuration facilitates incorporation into a microscope such as an inverted microscope or an upright microscope.

The sample observation method of the present invention includes the steps of: irradiating the sample with excitation light including a wavelength that excites the sample; guiding detection light emitted from the sample along with irradiation of the excitation light; and detecting the detection light. and the step of irradiating the sample with excitation light comprises: outputting light whose wavelength changes with time as excitation light from a wavelength swept light source; a step of emitting the excitation light from the spectroscopic element at an emission angle corresponding to the wavelength of the excitation light; a step of causing the excitation light emitted from the spectroscopic element to enter the cylindrical lens at an incidence angle corresponding to the emission angle; a step of condensing the excitation light guided by a relay optical system including a lens, and irradiating the sample with the sheet-like excitation light.

In this sample observation method, light whose wavelength changes with time is output from the wavelength swept light source as excitation light, the excitation light output from the wavelength swept light source is emitted from the spectroscopic element at an emission angle corresponding to the wavelength, and is emitted from the spectroscopic element. The emitted excitation light enters the cylindrical lens at an incident angle corresponding to the emitted angle. Thereby, the irradiation position of the sheet-like excitation light can be scanned with respect to the sample. As a result, since it is not necessary to move or rotate the sample during observation, it is possible to suppress the sample from shaking and to observe the sample stably. In addition, since the wavelength swept light source, spectroscopic element, and cylindrical lens are used to scan the irradiation position of the excitation light on the sample, the scanning speed of the excitation light on the sample can be increased. Therefore, according to this light sheet microscope, high-speed and stable observation can be realized.

According to the present invention, it is possible to provide a light sheet microscope and sample observation method capable of achieving high-speed and stable observation.

It is a figure which shows the structure of the light sheet microscope which concerns on embodiment. It is a figure for demonstrating a spectroscopic element. It is a figure which shows the irradiation optical system at the time of seeing from an X-axis direction. (a) and (b) are diagrams showing the irradiation optical system when viewed from the Y-axis direction. (a) to (c) are diagrams for explaining the adjustment of the beam waist position. (a) and (b) are diagrams showing the configuration around a container in which a sample is placed. (a) to (c) are diagrams showing modifications. It is a figure which shows another modification.

An embodiment of the present invention will be described in detail below with reference to the drawings. In the following description, the same reference numerals are used for the same or corresponding elements, and overlapping descriptions are omitted.

The light sheet microscope 1 shown in FIG. 1 irradiates a sample S with a sheet-like (planar) excitation light L1, and detects the detection light L2 emitted from the sample S along with the irradiation of the excitation light L1. It is an apparatus for acquiring an image of a sample S. In the light sheet microscope 1, the irradiation position of the excitation light L1 is scanned with respect to the sample S, and an image of the sample S is acquired at each irradiation position. In the light sheet microscope 1, since the region where the sample S is irradiated with the excitation light L1 is narrow, deterioration of the sample S such as photobleaching or phototoxicity can be suppressed, and image acquisition can be speeded up. can.

The sample S is, for example, a cell or living body sample containing a fluorescent substance such as a fluorescent dye or a fluorescent gene. The sample S emits detection light L2 such as fluorescence when irradiated with light in a predetermined wavelength range. The sample S is placed, for example, in a container 5 that is transparent to at least the excitation light L1 and the detection light L2. Details of the container 5 will be described later.

As shown in FIG. 1, the light sheet microscope 1 includes an illumination optical system 10, a detection optical system 20, a photodetector 30, and a controller 40. The irradiation optical system 10 irradiates the sample S with the excitation light L1. The detection optical system 20 guides the detection light L2 emitted from the sample S along with the irradiation of the excitation light L1 to the photodetector 30 . The photodetector 30 detects the detection light L2 guided by the detection optical system 20 . The control unit 40 controls operations of the irradiation optical system 10, the detection optical system 20, the photodetector 30, and the like.

The irradiation optical system 10 has a swept wavelength light source 11 , a spectral element 12 , a relay optical system 14 and a first objective lens 15 . The relay optical system 14 includes a cylindrical lens 16 and a lens 17.

The wavelength swept light source 11 outputs excitation light L1 including a wavelength that excites the sample S. As shown in FIG. The wavelength swept light source 11 outputs light whose wavelength changes with time as excitation light L1. More specifically, the wavelength swept light source 11 is a light source that performs wavelength sweeping within a predetermined wavelength range by periodically changing the wavelength of the output pumping light L1 at high speed.

Although the wavelength swept light source 11 may be any light source, it is preferable that the emission angle does not change when the wavelength is swept. As such a wavelength swept light source 11, for example, there is a semiconductor laser light source whose output light wavelength is variable according to changes in cavity length, temperature, or current value. The wavelength swept light source 11 may be a unit that combines a light source that outputs white laser light and an AOTF (Acousto-Optic Tunable Filter) that selectively transmits light in a specific wavelength range. Alternatively, the wavelength swept light source 11 is a Littrow external cavity semiconductor laser light source, a Vertical Cavity Surface Emitting Laser (VCSEL) light source, or an external cavity semiconductor laser light source using a KTN crystal. may be

The wavelength swept light source 11 may emit coherent light. As a laser light source that outputs coherent light, a light source that oscillates continuous waves may be used, a light source that oscillates pulse light such as ultrashort pulse light, or a light source that outputs intensity-modulated light may be used. may be used. Furthermore, a unit combining these light sources with an optical shutter or an AOM (Acousto-Optic Modulator) for pulse modulation may be used.

As shown in FIGS. 1 and 2, the excitation light L1 output from the wavelength swept light source 11 is incident on the spectroscopic element 12 . The spectral element 12 emits the excitation light L1 at an emission angle θ corresponding to the wavelength of the incident excitation light L1. In this embodiment, the spectral element 12 is a reflective blazed diffraction grating that diffracts the excitation light L1 at an output angle θ corresponding to the wavelength of the incident excitation light L1. The exit angle θ is, for example, the angle of the optical axis of the excitation light L1 emitted from the spectroscopic element 12 with respect to the normal line N of the grating surface 12a of the spectroscopic element 12 . FIG. 2 illustrates three excitation lights L1 having wavelengths λ1, λ2, and λ3, respectively, and emitted from the spectroscopic element 12 at different emission angles θ.

The excitation light L1 emitted from the spectral element 12 enters the cylindrical lens 16 at an incident angle corresponding to the emission angle θ. The spectroscopic element 12 is arranged, for example, so that when the excitation light L1 of the central wavelength in the wavelength sweeping by the wavelength swept light source 11 is emitted from the spectroscopic element 12, the excitation light L1 travels along the optical axis of the cylindrical lens 16. It is The cylindrical lens 16 is arranged on its optical axis at a distance (optical distance) equal to the focal length of the cylindrical lens 16 from the grating surface 12a (output surface) of the spectroscopic element 12 . The excitation light L<b>1 incident on the cylindrical lens 16 is guided to the first objective lens 15 via the lens 17 . The first objective lens 15 collects the excitation light L1 guided by the relay optical system 14 and irradiates the sample S with the excitation light L1 in the form of a sheet. The first objective lens 15 and lens 17 are arranged on the optical axis of the cylindrical lens 16 . Note that the spectral elements 12 do not necessarily have to be arranged as described above.

How the excitation light L1 is shaped into a sheet by the irradiation optical system 10 will be described with reference to FIGS. 3 and 4. FIG. The surface of the cylindrical lens 16 is curved along one direction (hereinafter referred to as the X-axis direction) perpendicular to its optical axis C, while the surface is curved in a direction orthogonal to the optical axis C and the X-axis direction (hereinafter referred to as the Y-axis direction). direction) is not curved but straight. 3 is a diagram showing the irradiation optical system 10 when viewed from the X-axis direction, and FIGS. 4A and 4B are diagrams showing the irradiation optical system 10 when viewed from the Y-axis direction. is.

As shown in FIG. 3, the cylindrical lens 16 does not function as a lens for the Y-axis component of the excitation light L1 output from the spectroscopic element 12 . When viewed from the X-axis direction, the excitation light L1 transmitted through the cylindrical lens 16 is condensed by the lens 17 onto the pupil of the first objective lens 15 . The lens 17 is, for example, a convex lens such as a biconvex lens or a plano-convex lens. As a result, the first objective lens 15 emits a strip-shaped excitation light L1 having a constant width (width in the Y-axis direction) W. As shown in FIG. This width W is the width W of the sheet-like excitation light L1 with which the sample S is irradiated. The width W of the sheet-shaped excitation light L1 can be adjusted by the focal length of the lens 17. FIG. Alternatively, the width W of the sheet-shaped excitation light L1 may be adjusted by inserting an aperture in the optical path between the wavelength swept light source 11 and the spectral element 12 to change the beam diameter, or by adjusting the focal length of the lens 17. may be adjusted by changing the

As shown in FIGS. 4A and 4B, the cylindrical lens 16 functions as a lens for the X-axis component of the excitation light L1 output from the spectral element 12. As shown in FIGS. When viewed from the Y-axis direction, the cylindrical lens 16 and the lens 17 constitute a pupil transmission optical system, and the excitation light L1 incident on the cylindrical lens 16 is transmitted to the pupil of the first objective lens 15 via the lens 17. be done. As a result, linear excitation light L1 parallel to the optical axis C is emitted from the first objective lens 15 . Therefore, when viewed three-dimensionally, the excitation light L1 emitted from the first objective lens 15 has a sheet shape.

As shown in FIG. 4B, the exit angle θ described above is the exit angle of the excitation light L1 from the spectroscopic element 12 when viewed from the Y-axis direction. That is, the spectroscopic element 12 emits the excitation light L1 at an emission angle θ corresponding to the wavelength of the excitation light L1 when viewed from the Y-axis direction.

The height (distance from the optical axis C in the X-axis direction) H of the sheet-shaped excitation light L1 emitted from the first objective lens 15 corresponds to the incident angle of the excitation light L1 incident on the cylindrical lens 16 . This is because the relay optical system 14 brings the pupil plane of the first objective lens 15 into a conjugate relationship with the lattice plane 12a. That is, the height H corresponds to the emission angle θ of the excitation light L1 emitted from the spectral element 12. FIG. In other words, in the irradiation optical system 10 , the output angle θ is converted into the height H of the sheet-shaped excitation light L<b>1 output from the first objective lens 15 . Therefore, by changing the wavelength of the excitation light L1 output from the wavelength swept light source 11 at high speed and changing the emission angle θ of the excitation light L1 emitted from the spectroscopic element 12 at high speed, the light emitted from the first objective lens 15 It is possible to change the height H of the sheet-shaped excitation light L1 to be emitted at high speed.

5(a) to 5(c), the beam waist of the excitation light L1 emitted from the first objective lens 15 (when viewed from the Y-axis direction, the thickness of the sheet-like excitation light is the smallest) The adjustment of the position of B) will be described. The sample S is irradiated with the excitation light L1 at the beam waist B as a sheet-like excitation light L1. That is, the position of the beam waist B corresponds to the formation position of the sheet-like excitation light L1 in the irradiation direction of the excitation light L1 with respect to the sample S.

As a means for adjusting the position of the beam waist B, the light sheet microscope 1 maintains the positional relationship (optical distance) between the spectroscopic element 12 and the cylindrical lens 16, while the wavelength swept light source 11, the spectroscopic element 12 and A moving mechanism 3 for moving the cylindrical lens 16 along the optical axis C is further provided. The moving mechanism 3 is, for example, a movable stage. The moving mechanism 3 is electrically connected to the control section 40 and its drive is controlled by the control section 40 .

As shown in FIGS. 5A and 5B, the moving mechanism 3 moves the wavelength swept light source 11, the spectroscopic element 12, and the cylindrical lens 16 away from the lens 17, thereby moving the beam waist B to the second 1 can be moved to the objective lens 15 side. As shown in FIGS. 5A and 5C, the moving mechanism 3 moves the wavelength swept light source 11, the spectral element 12, and the cylindrical lens 16 closer to the lens 17, thereby changing the beam waist B to the second 1 It can be moved to the side opposite to the objective lens 15 . The swept wavelength light source 11 may be optically connected to the spectroscopic element 12 via an optical member such as a fiber or a collimator lens. It is moved integrally with the spectral element 12 and the cylindrical lens 16 .

As shown in FIGS. 6( a ) and 6 ( b ), the sample S is placed inside the container 5 . The container 5 is made of, for example, glass or plastic, and has a bottom wall portion 51 and side wall portions 52 . The bottom wall portion 51 is, for example, a cover glass or a glass bottom dish. The side wall portion 52 extends, for example, from the edge portion of the bottom wall portion 51 along a direction perpendicular to the bottom wall portion 51, and has a cylindrical or square tube shape. The container 5 is arranged, for example, such that the bottom wall portion 51 is located vertically below the side wall portion 52 . The sample S is arranged, for example, together with the culture solution in a storage space defined by the bottom wall portion 51 and the side wall portion 52 . A sample S is placed on the bottom wall portion 51 . Note that when the sample is a dried product, the container 5 may not be used.

As shown in FIG. 6A, the first objective lens 15 is, for example, a water immersion objective lens, and is arranged so as to be submerged in the culture solution. A bottom wall portion 51 of the container 5 is provided with a mirror (reflection portion) 53 that reflects the excitation light L1 emitted from the first objective lens 15 toward the sample S at a predetermined angle (for example, perpendicularly). The mirror 53 has a reflecting surface 54a that extends at an angle of 45 degrees with respect to the optical axis of the first objective lens 15 and the bottom wall portion 51, respectively. The scanning direction of the sheet-like excitation light L1 with respect to the sample S is the direction indicated by the arrow A (the direction along the optical axis (optical axis C) of the first objective lens 15). Note that the mirror (reflector) 53 may not be provided on the container 5 . For example, the mirror 53 may be attached to the first objective lens 15 via an attachment.

As shown in FIG. 6(b), the first objective lens 15 may be a dry objective lens and may be placed outside the culture solution. The working distance of the dry objective is longer than that of the water immersion objective. In this case, the bottom wall portion 51 of the container 5 is provided with a prism (reflection portion) 54 that reflects the excitation light L1 emitted from the first objective lens 15 toward the sample S at a predetermined angle (for example, perpendicularly). . The prism 54 has a reflective surface 54 a extending at an angle of 45 degrees with respect to the optical axis of the first objective lens 15 and the bottom wall portion 51 . The scanning direction of the sheet-shaped excitation light L1 with respect to the sample S is the direction indicated by the arrow A. As shown in FIG. Note that the prism (reflecting portion) 54 may not be provided on the container 5 . For example, the prism 54 may be attached to the first objective lens 15 via an attachment.

If the reflector is the mirror 53, a water immersion objective lens with a short working distance can be used. In this case, the same medium exists between the first objective lens 15 and the sample S, and there is no need to correct aberrations that occur when passing through the interface of different types of media. number (NA) can be increased. On the other hand, if the reflector is a prism 54, a dry objective lens with a long working distance can be used. In this case, the trouble of cleaning the lens can be saved, and measurements such as frequently exchanging the sample S, measurements using a liquid that invades the first objective lens 15, and long-term measurements can be easily performed. can be done.

The detection optical system 20 has a second objective lens 21 and a liquid lens (focus position adjuster) 22 . The second objective lens 21 guides the detection light L2 emitted from the sample S along with the irradiation of the excitation light L1 to the photodetector 30 side. The second objective lens 21 is arranged to face the sample S with the bottom wall portion 51 interposed therebetween. As shown in FIGS. 1, 6(a) and 6(b), the optical axis of the second objective lens 21 is parallel to the optical axis of the first objective lens 15. It is perpendicular to the plane on which the shaped excitation light L1 is formed. In this embodiment, the excitation light L1 that is emitted from the first objective lens 15 and travels downward in the vertical direction is reflected by the mirror 53 or the prism 54, travels in the horizontal direction, and irradiates the sample S. A detection light L2 emitted from the sample S along with irradiation of the excitation light L1 and traveling downward in the vertical direction is incident on the second objective lens 21 .

The liquid lens 22 is a lens whose focal length is variable according to an input signal. In the detection optical system 20 , the focal position of the second objective lens 21 can be adjusted by changing the focal length of the liquid lens 22 . In the light sheet microscope 1, synchronizing with the change in the wavelength of the excitation light L1 output from the wavelength swept light source 11 so that the focal position of the second objective lens 21 coincides with the irradiation position of the excitation light L1 on the sample S, The focal length of the liquid lens 22 changes. This allows the detection light L2 to form an image on the photodetector 30 . Therefore, even when the irradiation position of the excitation light L1 on the sample S is scanned at high speed, the detection light L2 can be detected with high accuracy. The detection optical system 20 may further have a convex lens arranged between the second objective lens 21 and the liquid lens 22 . In this case, the adjustment range of the focal position of the second objective lens 21 by the liquid lens 22 can be expanded.

The photodetector 30 captures an image of the detection light L2 guided by the second objective lens 21 . Examples of the photodetector 30 include a CMOS camera, a CCD camera, a multi-anode photomultiplier tube, a two-dimensional image sensor such as SPAD (Single Photon Avalanche Diode), or a line sensor. Alternatively, photodetector 30 may be a point photosensor, such as an avalanche photodiode, or a spectrometer.

The control unit 40 is configured by, for example, a computer including a processor, memory, and the like. The control unit 40 controls the operation of the moving mechanism 3, the wavelength swept light source 11, the liquid lens 22, the photodetector 30, etc. by the processor, and executes various controls. For example, the control unit 40 changes the wavelength of the excitation light L1 output from the wavelength swept light source 11 over time so that the emission angle θ of the excitation light L1 emitted from the spectroscopic element 12 changes over time. In addition, the control unit 40 synchronizes with the change in the wavelength of the excitation light L1 output from the wavelength swept light source 11 so that the focal position of the second objective lens 21 coincides with the irradiation position of the excitation light L1 on the sample S. , changes the focal length of the liquid lens 22 . At least one of the first objective lens 15 and the second objective lens 21 may be movable along its optical axis by a driving element such as a piezo actuator or a stepping motor. In this case, the control unit 40 also controls the operation of the driving element.

As described above, in the light sheet microscope 1, light whose wavelength changes with time is output from the wavelength swept light source 11 as the excitation light L1, and the excitation light L1 output from the wavelength swept light source 11 has an emission angle corresponding to the wavelength. The excitation light L1 emitted from the spectroscopic element 12 at θ enters the cylindrical lens 16 at an incident angle corresponding to the emission angle θ. Thereby, the irradiation position of the sheet-like excitation light L1 can be scanned with respect to the sample S. As a result, since it is not necessary to move or rotate the sample S during observation, it is possible to suppress the sample S from shaking and to observe the sample S stably. Further, since the irradiation position of the sample S with the excitation light L1 is scanned using the wavelength swept light source 11, the spectroscopic element 12, and the cylindrical lens 16, the scanning of the sample S with the excitation light L1 can be sped up. Therefore, according to the light sheet microscope 1, high-speed and stable observation can be realized. In addition, compared to the case of mechanically scanning the irradiation position of the sheet-shaped excitation light L1 on the sample S using, for example, a galvanomirror, etc., the stability is excellent, the life of the parts is long, and the speed can be further increased. can be planned. Furthermore, the center wavelength and wavelength sweep range of the excitation light L1 with which the sample S is irradiated can be changed over time. Therefore, it can be suitably used for observation that requires changing the center wavelength and wavelength sweep range of the irradiation light to the sample S with time. For example, when the chemical composition of the object to be measured changes with time and the peak of the absorption spectrum changes, the center wavelength and wavelength sweep range of the irradiation light can be corrected (calibrated) according to the change. Alternatively, when the observation target itself changes with time, the central wavelength and wavelength sweep range of the irradiation light can be changed to the central wavelength and wavelength sweep range suitable for the absorption spectrum of the observation target substance.

In the light sheet microscope 1, the spectral element 12 is a diffraction grating. As a result, the above operational effect of being able to realize high-speed and stable observation can be remarkably achieved.

In the light sheet microscope 1, the detection optical system 20 synchronizes with changes in the wavelength of the excitation light L1 output from the wavelength swept light source 11 and includes a liquid lens 22 (focus position adjustment lens 22) that changes the focal position of the second objective lens 21. vessel). Thereby, even when the irradiation position of the excitation light L1 on the sample S is scanned at high speed, the detection light L2 can be detected with high accuracy.

The light sheet microscope 1 includes a moving mechanism 3 that moves the swept wavelength light source 11, the spectroscopic element 12, and the cylindrical lens 16 along the optical axis C while maintaining the positional relationship between the spectroscopic element 12 and the cylindrical lens 16. ing. Thereby, the position of the beam waist B of the excitation light L1 emitted from the first objective lens 15, that is, the formation position of the sheet-like excitation light L1 in the irradiation direction of the excitation light L1 to the sample S can be adjusted. When the reflecting portion is the prism 54, it is possible to suppress the displacement of the beam waist B due to the change in the optical path length by causing the excitation light L1 to pass through the prism 54. FIG.

In the light sheet microscope 1, the container 5 is provided with a mirror 53 or a prism 54 (reflector) that reflects the excitation light L1 emitted from the first objective lens 15 toward the sample S. As a result, the sample S can be irradiated with the sheet-shaped excitation light L<b>1 without being affected by the side wall portion 52 of the container 5 . In addition, the reflecting portion for guiding the excitation light L1 can be integrated with the container 5, so that the number of parts can be reduced and the size of the device can be reduced. In addition, compared to the case where the reflector is separate from the container 5, the sample S can be irradiated with the excitation light L1 reliably and accurately. In addition, the first objective lens 15 and the second objective lens 21 can be arranged so that their optical axes are parallel to each other, which facilitates incorporation into a microscope such as an inverted microscope or an erect microscope. Note that the mirror 53 or the prism 54 may not be provided on the container 5 . For example, the mirror 53 or prism 54 may be attached to the first objective lens 15 via an attachment. In other words, by converting the angle modulation by the spectral element 12 into height modulation and changing the traveling direction of the sheet-like excitation light L1, it is possible to facilitate incorporation into a general-purpose microscope. Further, by setting the reflection angle of the reflecting portion, the first objective lens 15 and the second objective lens 21 can be freely arranged three-dimensionally within a range in which they do not interfere with each other. In addition, it is possible to facilitate combination with microcell chambers or microchannels, and to realize reliable observation.

In the light sheet microscope 1 , the optical axis of the second objective lens 21 is parallel to the optical axis of the first objective lens 15 . This facilitates incorporation into a microscope such as an inverted microscope or an upright microscope.

A sample observation method using the light sheet microscope 1 includes the step of irradiating the sample S with excitation light L1 including a wavelength that excites the sample S, and guiding the detection light L2 emitted from the sample S along with the irradiation of the excitation light L1. and detecting the detection light L2. The step of irradiating the sample S with the excitation light L1 includes a step of outputting the light whose wavelength changes with time as the excitation light L1 from the wavelength swept light source 11, and a step of outputting the excitation light L1 output from the wavelength swept light source 11 to the spectroscopic element 12. and emits the excitation light L1 from the spectroscopic element 12 at an emission angle θ corresponding to the wavelength of the excitation light L1; and a step of condensing the excitation light L1 guided by the relay optical system 14 including the cylindrical lens 16 and irradiating the sample S with the sheet-like excitation light L1. According to this sample observation method, high-speed and stable observation can be realized for the reasons described above.

Although one embodiment of the present invention has been described above, the present invention is not limited to the above embodiment. For example, the material and shape of each configuration are not limited to the materials and shapes described above, and various materials and shapes can be adopted.

As shown in FIG. 7A, the spectral element 12 may be a transmission diffraction grating. Such a spectroscopic element 12 is configured with a slit, for example. As shown in FIG. 7(b), the spectral element 12 may be a prism. In this case, the spectroscopic element 12 is configured to be rotatable around the Y-axis direction, and the irradiation optical system 10 has a driving section that rotates the spectroscopic element 12 around the Y-axis. This drive unit is, for example, a stepping motor or a piezo actuator. The drive section is electrically connected to the control section 40 and controlled by the control section 40 . The spectroscopic element 12 is rotated around the Y-axis by the driving unit according to the wavelength of the excitation light L1 output from the wavelength swept light source 11 so that the excitation light L1 is emitted from the spectroscopic element 12 on the optical axis C. Let me. As shown in FIG. 7(c), the spectroscopic element 12 may be a grism in which a diffraction grating and a prism are combined. Even with these modified examples, high-speed and stable observation can be realized as in the case of the above-described embodiment. Further, when the spectroscopic element 12 is a grism, the traveling direction of the light of the center wavelength does not change before and after the incident light, so that the design of the optical system can be facilitated.

In the above embodiment, the wavelength swept light source 11 may be configured to be able to switch the central wavelength of the pumping light L1 among a plurality of central wavelengths. In this case, the spectroscopic element 12 is configured to be rotatable around the Y-axis direction, and the irradiation optical system 10 has a driving section that rotates the spectroscopic element 12 around the Y-axis. This drive unit is, for example, a stepping motor or a piezo actuator. The drive section is electrically connected to the control section 40 and controlled by the control section 40 . The spectroscopic element 12 is rotated around the Y-axis direction according to the central wavelength of the excitation light L1 output from the wavelength swept light source 11 by the drive unit so that the excitation light L1 having the central wavelength travels along the optical axis C. Let me. Even with this modification, high-speed and stable observation can be realized as in the case of the above-described embodiment. Moreover, the sample S can be irradiated with the excitation light L1 having a plurality of center wavelengths.

As shown in FIG. 8, the irradiation optical system 10 may have a plurality of wavelength swept light sources 11A and 11B that output lights having different center wavelengths as the excitation light L1. In this case, the excitation light L1 output from each of the wavelength swept light sources 11A and 11B is incident on the spectroscopic element 12 at an incident angle corresponding to its center wavelength. That is, the wavelength swept light sources 11A and 11B are arranged so that the incident condition is satisfied. Specifically, the excitation light L1A output from the wavelength swept light source 11A is emitted from the spectral element at an incident angle φ1 corresponding to the central wavelength of the excitation light L1A so that the excitation light L1A with the central wavelength travels along the optical axis C. 12. The excitation light L1B output from the wavelength swept light source 11B enters the spectroscopic element 12 at an incident angle φ2 corresponding to the center wavelength of the excitation light L1B so that the excitation light L1B with the center wavelength travels along the optical axis C. Incident angles φ1 and φ2 are different from each other. Even with this modification, high-speed and stable observation can be realized as in the case of the above-described embodiment. Moreover, the sample S can be irradiated with the excitation light L1 having a plurality of center wavelengths. In addition, in this modification, a mirror or the like having a variable reflection angle may be arranged between at least one of the plurality of swept wavelength light sources 11 and the spectroscopic element 12 . At least one of the plurality of wavelength swept light sources 11 may output the pumping light L1 to the spectroscopic element 12 via an optical fiber.

In the above embodiments, the liquid lens 22 may be omitted. In this case, the second objective lens 21 may be mechanically moved by a piezo actuator or the like, or the zoom lens arranged between the second objective lens 21 and the photodetector 30 may be mechanically moved. may However, in the above embodiment, since the focal position of the second objective lens 21 is changed at high speed by electrical control using the liquid lens 22 , the focal position of the second objective lens 21 is changed by the light emitted from the spectral element 12 . It is possible to reliably synchronize with the change in the emission angle θ of the excitation light L1. Alternatively, when the liquid lens 22 is omitted, an objective lens with a deep focal depth is used as the second objective lens 21, and the sheet-shaped excitation light L1 is scanned over the sample S within the focal depth of the second objective lens 21. may In this case, adjustment of the focal position of the second objective lens 21 can be omitted.

The cylindrical lens 16 may be composed of a spatial light modulator (SLM) that modulates the excitation light L1 according to a phase pattern corresponding to the cylindrical lens. The focal position adjuster only needs to be able to adjust the focal position of the second objective lens 21 , and may be composed of something other than the liquid lens 22 . The moving mechanism 3 may be omitted. Mirror 53 or prism 54 may be configured separately from container 5 . The optical axis of the first objective lens 15 and the optical axis of the second objective lens 21 may cross each other (for example, orthogonally). The detection optical system 20 separates the excitation light L1 and the detection light L2 from the light guided by the second objective lens 21, for example, between the second objective lens 21 and the liquid lens 22, and extracts the extracted detection light. It may further have an optical filter that outputs L2 to the photodetector 30 side. The spectral element 12 may be configured by a spatial light modulator. For example, the spectral element 12 may be configured by a spatial light modulator that modulates the excitation light L1 according to a diffraction grating pattern. In this case, the grating constant can be changed by changing the diffraction grating pattern. The cylindrical lens 16 and spectroscopic element 12 may be configured by one spatial light modulator.

DESCRIPTION OF SYMBOLS 1... Light sheet microscope, 3... Moving mechanism, 5... Container, 10... Irradiation optical system, 11... Wavelength swept light source, 12... Spectral element, 14... Relay optical system, 15... First objective lens, 16... Cylindrical lens, 20... Detection optical system, 21... Second objective lens, 22... Liquid lens (focal position adjuster), 30... Photodetector, 53... Mirror (reflection part), 54... Prism (reflection part), B... Beam waist , C... Optical axis, L1... Excitation light, L2... Detection light, S... Sample, ?... Output angle.

Claims (15)

an irradiation optical system that irradiates the sample with excitation light including a wavelength that excites the sample;
a detection optical system for guiding detection light emitted from the sample along with irradiation of the excitation light;
a photodetector that detects the detection light guided by the detection optical system,
The irradiation optical system is
a wavelength swept light source that outputs light whose wavelength changes with time as the excitation light;
a spectroscopic element that receives the excitation light output from the wavelength swept light source and emits the excitation light at an emission angle corresponding to the wavelength of the excitation light;
a relay optical system including a cylindrical lens into which the excitation light emitted from the spectroscopic element is incident at an incident angle corresponding to the emission angle;
a first objective lens for condensing the excitation light guided by the relay optical system and for irradiating the sample with the sheet-shaped excitation light. 2. The light sheet microscope according to claim 1, wherein said cylindrical lens is arranged on the optical axis of said cylindrical lens at a distance equal to the focal length of said cylindrical lens from the exit surface of said spectroscopic element. The spectroscopic element has an exit surface from which the excitation light incident from the swept wavelength light source exits,
The cylindrical lens constitutes the relay optical system, and the relay optical system receives the excitation light incident on the cylindrical lens, and the pupil of the first objective lens has a conjugate relationship with the exit surface of the spectroscopic element. 3. The light sheet microscope according to claim 1, wherein a pupil transmission optical system for transmission to the pupil of said first objective lens is constructed such that: 4. The light sheet microscope according to claim 1, wherein said spectral element is a diffraction grating. 4. The light sheet microscope according to claim 1, wherein said spectral element is a prism. The detection optical system includes a second objective lens into which the detection light is incident, and a focal point that changes the focal position of the second objective lens in synchronization with changes in the wavelength of the excitation light output from the wavelength swept light source. A light sheet microscope according to any one of claims 1 to 5, comprising a position adjuster. 7. A light sheet microscope according to claim 6, wherein said focus position adjuster is a liquid lens. The irradiation optical system has a plurality of the wavelength swept light sources,
wherein the plurality of wavelength swept light sources output light having different center wavelengths as the excitation light;
8. The light sheet microscope according to any one of claims 1 to 7, wherein said excitation light output from said plurality of wavelength swept light sources is incident on said spectroscopic element at an incident angle corresponding to its center wavelength. 3. A movement mechanism for moving said wavelength-swept light source, said spectroscopic element and said cylindrical lens along an optical axis of said cylindrical lens while maintaining a positional relationship between said spectroscopic element and said cylindrical lens. 9. The light sheet microscope according to any one of 1-8. The light sheet microscope according to any one of claims 1 to 9, further comprising a reflector that reflects the excitation light emitted from the first objective lens toward the sample. further comprising a container in which the sample is placed;
11. The light sheet microscope according to claim 10, wherein said container is provided with said reflector. The detection optical system has a second objective lens on which the detection light is incident,
The light sheet microscope according to any one of claims 1 to 11, wherein the optical axis of said second objective lens is parallel to the optical axis of said first objective lens. irradiating the sample with excitation light having a wavelength that excites the sample;
a step of guiding detection light emitted from the sample along with irradiation of the excitation light;
and detecting the detection light;
The step of irradiating the sample with the excitation light includes
a step of outputting light whose wavelength changes with time from a wavelength swept light source as the excitation light;
a step in which the excitation light output from the wavelength swept light source is incident on a spectroscopic element, and the excitation light is emitted from the spectroscopic element at an emission angle corresponding to the wavelength of the excitation light;
a step in which the excitation light emitted from the spectroscopic element is incident on a cylindrical lens at an incident angle corresponding to the emission angle;
and a step of condensing the excitation light guided by a relay optical system including the cylindrical lens by a first objective lens , and irradiating the sample with the sheet-like excitation light. 14. The sample observation method according to claim 13, wherein said cylindrical lens is arranged on the optical axis of said cylindrical lens at a distance equal to the focal length of said cylindrical lens from the exit surface of said spectroscopic element. The spectroscopic element has an exit surface from which the excitation light incident from the swept wavelength light source exits,
The cylindrical lens constitutes the relay optical system, and the relay optical system receives the excitation light incident on the cylindrical lens, and the pupil of the first objective lens has a conjugate relationship with the exit surface of the spectroscopic element. 15. The sample observation method according to claim 13, wherein a pupil transmission optical system for transmission to the pupil of said first objective lens is constructed so that .

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